Rett Syndrome (RTT) is a devastating disorder that afflicts the human Central Nervous System (CNS). Patients with RTT have loss of function mutations in the gene encoding Methyl-CpG Binding Protein 2 (MeCP2). Conversely patients with duplications covering the MECP2 locus have a distinct but overlapping disease, MECP2 Duplication Syndrome (MDS). These and other studies have demonstrated that proper control of MeCP2 levels within the mammalian CNS is vital for normal neuronal function. Post-Translational Modification (PTM) is known to control the abundance of many proteins, leading to the hypothesis that MeCP2 levels may also be controlled by certain forms of PTM. Preliminary data have identified two methyltransferases that alter MeCP2 stability. One methyltransferase decreases MeCP2 stability and requires a methylation site we have identified within MeCP2. The second methyltransferase increases MeCP2 stability. In order to better understand the role of methylation in modulating MeCP2 levels, previously derived genetically engineered mice will be used to alter the expression of these two enzymes. These mice will be assessed for changes in functional MeCP2 abundance. Mouse models expressing low levels of MeCP2 or high levels of MeCP2 both have well characterized phenotypes. These Mecp2 mutant mice will be mated to mice engineered to bear mutations in the potential enzymatic modifiers. Resulting double mutant mice will be evaluated for a genetic interaction between Mecp2 and the identified methyltransferases. These studies will elucidate whether loss of function of these enzymes can alleviate symptoms in mouse models of RTT and MDS. In a second aim, additional methylation sites within the MeCP2 polypeptide will be identified using tandem mass spectrometry. This information will be used to generate specific antibodies that recognize modified forms of MeCP2. These antibodies will be used to confirm the existence and importance of these methylation sites in vivo. Finally small molecular inhibitors of relevant enzymes will be tested for the ability to alter MeCP2 levels in cell culture. These studies will form a foundation for later drug studies towards the treatment of patients with disorders of MeCP2 levels. MeCP2 is required at precise physiological levels for normal neuronal function. There are currently no specific pharmacological treatments for either Rett Syndrome or MECP2 Duplication Syndrome. PUBLIC HEALTH RELEVANCE: These studies will explore the potential of treating these disorders using drugs which target certain forms of Post- Translational Modification and subsequently alter functional MeCP2 abundance.